Francisco J. Méndez

The conserved undecapeptide shared by thiol‐activated cytolysins is involved in membrane binding

Thiol‐activated cytolysins share a conserved hydrophobic, Trp‐rich undecapeptide that is suggested to be involved in membrane binding and intercalation. The neutralizing monoclonal antibody PLY‐5 recognizes all members of this toxin family and peptide mapping assigned its epitope to the undecapeptide motif. This antibody inhibited binding of the toxins to host cell membranes and the epitope was no longer available for binding when a preformed toxin/membrane complex was tested. These results confirm the model of cytolysin binding suggested by structural data.

The role of pneumolysin in mediating lung damage in a lethal pneumococcal pneumonia murine model

BackgroundIntranasal inoculation of Streptococcus pneumoniae D39 serotype 2 causes fatal pneumonia in mice. The cytotoxic and inflammatory properties of pneumolysin (PLY) have been implicated in the pathogenesis of pneumococcal pneumonia.MethodsTo examine the role of PLY in this experimental model we performed ELISA assays for PLY quantification. The distribution patterns of PLY and apoptosis were established by immunohistochemical detection of PLY, caspase-9 activity and TUNEL assay on tissue sections from mice lungs at various times, and the results were quantified with image analysis. Inflammatory and apoptotic cells were also quantified on lung tissue sections from antibody treated mice.ResultsIn bronchoalveolar lavages (BAL), total PLY was found at sublytic concentrations which were located in alveolar macrophages and leukocytes. The bronchoalveolar epithelium was PLY-positive, while the vascular endothelium was not PLY reactive. The pattern and extension of cellular apoptosis was similar. Anti-PLY antibody treatment decreased the lung damage and the number of apoptotic and inflammatory cells in lung tissues.ConclusionThe data strongly suggest that in vivo lung injury could be due to the pro-apoptotic and pro-inflammatory activity of PLY, rather than its cytotoxic activity. PLY at sublytic concentrations induces lethal inflammation in lung tissues and is involved in host cell apoptosis, whose effects are important to pathogen survival.

Streptococcus pneumoniae virulence factors and their clinical impact: an update

The morbidity and mortality rates associated with Streptococcus pneumoniae remain very high worldwide. The virulence of this bacterium is largely dependent on its polysaccharide capsule, which is quite heterogeneous and represents a serious obstacle for designing effective vaccines. However, it has been demonstrated that numerous protein virulence factors are involved in the pathogenesis of pneumococcal disease. An important related finding from experimental animal models is that non-capsulated strains of pneumococci are protective against capsulated ones. Hence, new vaccine designs are focused on the surface proteins (e. g., PspA and PspC) and on the cytolysin, pneumolysin. Moreover, several virulence factors have potential value for pneumococcal diagnosis by urinalysis. In this paper, we review the virulence factors involved in bacteria-host interactions, and the new developments in vaccines and diagnostic methods.

A specific and ultrasensitive chemiluminescent sandwich ELISA test for the detection and quantitation of pneumolysin.

A chemiluminescent sandwich ELISA test has been developed for the detection and quantitation of pneumolysin. The test is based on a mouse monoclonal as the capture antibody and on rabbit polyclonal IgGs as detection antibodies, in combination with an anti-rabbit IgG alkaline phosphatase conjugate. The estimated detection limit of the purified recombinant toxin in phosphate-buffered saline with 0.05% Triton X-100 is around 5 pg ml−1, with averaged intra- and inter-assay variation coefficients of 7% and 13.5%, respectively. The assay has been applied to the quantitation of pneumolysin in pneumococcal isolates, providing, for the first time, a direct measurement of the amount of the toxin produced by different strains; a variation has been found in their pneumolysin content. The test is highly specific as no other purified toxins or human pneumonia- or meningitis-associated bacteria yielded false-positive results. This specific and highly sensitive method could help in the diagnosis of human infections.

Survey of Escherichia coli septicemia over a six-year period.

Escherichia coli was the most frequent species isolated from blood cultures in the Hospital Covadonga of Oviedo (Spain) over a six-year period (474 episodes, 15.3 % of the total septicemias and 2.7 episodes per 1,000 patients).Escherichia coli strains were susceptible in >95 % of episodes to cefoxitin, cefotaxime, gentamicin, tobramicin and amikacin. In a series of 72 episodes, microbiological features and host factors were studied. No endemic strains were found. Type 1 fimbria was detected in 73.6% of strains and P-fimbriae in 12.5 %, without correlation between P-fimbria and urinary infection; 84.7 % of the strains were resistant to decomplement human serum; 61.1 % produced aerobactin and 20.8 % were hemolytic. Factors such as age, hospital location, metastatic focus and surgical treatment were significantly correlated with morbidity and mortality. The global mortality rate was 18 %, and in 8.3 % of cases was directly associated with septicemia.

Characterisation of mouse monoclonal antibodies for pneumolysin: fine epitope mapping and V gene usage

Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) produced by Streptococcus pneumoniae, the main cause of community-acquired pneumonia. We have applied a set of diverse molecular methodologies (PCR-derived PLY peptides, biopanning of a library of phage-displayed random nonapeptides, indirect ELISA and competition tests with soluble peptides) to achieve concordant complementary observations in order to obtain a fine epitope mapping of three mouse monoclonal antibodies (PLY-4, PLY-7 and PLY-8) for PLY. PLY-4 seems to recognise a conformation-dependent epitope with a core reactivity involving R232. The epitopes recognised by PLY-7 and PLY-8 are within the sequences (401)GQDLTAH(407) and (450)KRTISIWGT(458), respectively. PLY-7 also recognises suilysin (SLY), in which the homologous reactive amino acid stretch is (429)GVNLTSH(435). In a homology model of PLY with the crystal structure of perfringolysin O (PFO), R232 is part of a well-exposed contorted loop on the edge of the concave and convex faces of domain 1. The sequences reactive with PLY-7 and PLY-8 would conform one of the loops at the bottom of domain 4 and a beta strand of one of the two beta sheets of this domain, respectively. Western blot analyses carried out with anti-PLY rabbit IgG and polyclonal mouse serum identified stretches comprising residues 40-98, 199-248, 352-414 and 415-471 of PLY as immunogenic and antigenic; altogether with their recognition by the monoclonal antibodies herein considered, these results stress the immunological significance of domains 1 and 4 of the PLY molecule. PLY-4, PLY-7 and PLY-8 share the same Vkappa chain; this chain and that of the PLY-5 monoclonal antibody are essentially in germline configuration, whereas the VH regions of these monoclonals come from diverse gene segments and are mutated.

Survey of bacteraemia in a Spanish hospital over a decade (1981-1990).

The aetiology, epidemiology and in-vitro antibiotic susceptibility of bacteraemic isolates in a Spanish hospital, over a decade (1981-1990), were reviewed. A total of 4363 possible episodes were microbiologically confirmed (incidence: 15.7 cases per 1000 admissions). A slight increase in the number of Gram-positive cocci and anaerobes over the last 4 years was found. The most frequent species (annual range) were: Escherichia coli (13.5%-18.9%), Staphylococcus aureus (10.5%-17.8%), Klebsiella spp. (3.0%-9.4%), Enterococcus faecalis (3.0%-8.2%), Pseudomonas aeruginosa (2.5%-5.8%) and Streptococcus pneumoniae (1.8%-5.6%). Males were more affected than females (ratio 1.7:1). The portal of entry was known in 2126 episodes, of which the most frequent were urinary and gasto-intestinal tracts (24.4% and 19.4% respectively). Bacteraemia was associated with rapidly and ultimately fatal underlying diseases in 7.2% and 15.1% of the cases respectively. The hospital location of patients was: medical ward 46.0%, surgical ward 22.7%, paediatric ward 16.9%, and intensive care unit 14.4%.

Susceptibility of Candida species isolated from female prostitutes with vulvovaginitis to antifungal agents and boric acid.

Abstract The aim of this study was to determine the antifungal susceptibility of 108 Candida albicans and 23 Candida glabrata isolates obtained from female prostitutes with vulvovaginitis, a population for which available data is limited. Amphotericin B, flucytosine, and fluconazole were tested by broth microdilution, and boric acid was tested by the agar dilution method. The susceptibility patterns found in this population were the same as those in the general population. Candida glabrata required greater concentrations of boric acid for inhibition in vitro than did Candida albicans.

Performance of a Pneumolysin Enzyme-Linked Immunosorbent Assay for Diagnosis of Pneumococcal Infections

ABSTRACT A pneumolysin-specific enzyme-linked immunosorbent assay (PLY-ELISA) for the detection of pneumolysin in urine was developed and evaluated in comparison with the commercially available Binax Now Streptococcus pneumoniae test (Binax, Portland, ME) for the diagnosis of pneumococcal infections. Assay sensitivity was evaluated using urine from 108 patients with culture-confirmed pneumococcal infections. In adults, the sensitivity and specificity of the PLY-ELISA were 56.6% and 92.2%, respectively. In children with nasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 62.5% and 87.5%, respectively, while specificities were 94.4% and 27.8%, respectively. In children with nonnasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 68.7% and 93.7%, respectively, and test specificities were 94.1% and 41.2%, respectively. The persistence of pneumolysin in urine of pneumococcal pneumonia patients decreased significantly after 4 to 6 days of treatment. Our data suggest that combining the high specificity of the PLY-ELISA with the high sensitivity of the Binax Now S. pneumoniae test would enable pneumococcal infections to be accurately diagnosed in children.

Characteristics ofPseudomonas aeruginosa strains causing septicemia in a Spanish hospital 1981–1990

The epidemiological and biochemical characteristics ofPseudomonas aeruginosa strains causing septicemia in a Spanish hospital over a ten-year period (1981–1990) were analyzed. A total of 207 episodes, corresponding to 0.7 episodes per 1,000 inpatients and 3.2 % of the total number of episodes of septicemia, were registered. Males were more often affected than females (rate 3.2:1). The respiratory (24.6 %) and urinary (21.2 %) tracts were the main portals of entry, while haematologic and solid tumours (15.4 %) were the most frequent underlying diseases. More than 86 % of the strains were susceptible to ceftazidime, mezlocillin, piperacillin and amikacin. Seventy strains were subjected to typing and analysis of virulence factors. Serotypes O:6, O:11 and O:2 could be considered endemic (each present in more than 11.4 % of strains). Pyocin typing, antibiotyping and resistotyping were preferred as secondary typing methods to phage typing and plasmid profile analysis. The combination of methods revealed a large diversity of strains although some cluster predominated. More than 80 % of the strains produced several exoenzymes, possessed pyoverdin and showed haemolytic activity, and all except one showed serum resistance. All strains were susceptible to silver and more than 80 % to mercury and boron, but all were resistant to iodine.